Inhibition of biosynthesis of triglycerides by certain N-β-phenethyl-N-pyridylalkylamines

ABSTRACT

Biosynthesis of triglycerides is inhibited by certain N-β-phenethyl-N-pyridylalkylamines.

This application is a continuation-in-part of application Ser. No.117,160, filed on Jan. 31, 1980, now abandoned.

DESCRIPTION OF THE INVENTION

It has been found that biosynthesis of triglycerides in swine isinhibited by certain N-β-phenethyl-N-pyridylalkylamines, of the generalformula: ##STR1## wherein (a) the bridging fragment >A--B-- is one of##STR2## and >C═CH--; (b) R is 2-pyridyl or ##STR3## (c) each of R¹ ishydrogen or methyl; (d) R² is hydrogen or hydroxyl;

(e) n is zero or one;

(f) X is halogen or alkyl of one or two carbon atoms;

(g) m is zero or one;

(h) Y is alkyl of one to three carbon atoms,

and their physiologically acceptable acid addition salts.

Suitable salts are those of such acids as acetic, succinic, maleic,fumaric, propionic, citric, lactic, hydrochloric, sulfuric andphosphoric acids.

Included in the invention are the individual optically active isomers,and diastereomers, as well as mixtures thereof, that inhibit synthesisof triglycerides.

The compounds of Formula I are known: they, and a method for theirpreparation, are described in U.S. Pat. No. 3,426,034.

The compounds of Formula I have been found to inhibit synthesis oftriglycerides in tissues of swine. The manner in which they cause thiseffect has not been established. Their effectiveness for this purposehas been ascertained by the following procedure.

An enzyme was prepared by homogenizing one gram of slices from pigadipose tissue, each slice being approximately 0.3 mm thick, inonemilliliter portions of a liquid medium (Liquid A). The homogenatethen was centrifuged (10.000 g×15 minutes), and the supernatant phase(the enzyme) was poured through cheesecloth to remove fat that had risento the top of the centrifuge tubes.

Liquid A had the following composition (in water):

0.15 M potassium chloride, 1 mM ethylenediaminetetracetic acid, 10 nMHepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 1 mM DTT(dithiothreitol), pH=7.0.

A solution of the test compound (Liquid B) was prepared: 10 milligramsof test compound plus 0.5 milliliter dimethylsulfoxide (DMSO) plus 1.83milliliters of glass-distilled water.

A premix (Liquid C) was freshly prepared before each test, from thefollowing components:

    ______________________________________                                                                     A-                                               Solutions                    mount                                            (in water)                   (ml)                                             ______________________________________                                        1 M Hepes (pH = 7.4)         3.00                                             0.2 M adenosine triphosphate (ATP) (pH = 7.0)                                                              0.30                                             10 mM coenzyme A (pH = 6.6, containing 15 mM DTT)                                                          0.16                                             0.1 M DTT (pH = 7.0)         0.20                                             5% fatty acid-free bovine serum albumin                                                                    0.20                                             1 M magnesium chloride       0.10                                             Water                        0.44                                             20 mM potassium palmitate (kept warm and added to the                         other components just before use of the mixture)                                                           0.60                                             ______________________________________                                    

0.163 milliliter of Liquid B (containing the test compound, or forcomparison omitting the test compound) was mixed with 0.1 milliliter ofLiquid C, 0.05 milliliter of a solution of L-[U-¹⁴ C]glycerol-3-phosphate (containing 5 microcuries of ¹⁴ C per milliliter),0.01milliliter of 0.8 M potassium phosphate (pH=7.4), 0.07 milliliter of0.2 M glycerol-3-phosphate (pH=7.4) and 0.047 milliliter of water.

0.25 milliliter of enzyme then was added. The final concentrations ofDMSO and test compound were 5% and 1000 micrograms per milliliter,respectively. The mixture was incubated for 8 minutes at 37° C. A blankcontrol without enzyme was incubated under the same conditions. Then, 3milliliters of a 1:2 (v/v) mixture of chloroform and methanol was addedand thoroughly mixed to terminate the reaction. After 10 minutes, 2milliliters of chloroform and 1 milliliter of 0.1 N hydrochloric acidwere added to the stirred mixture. The mixture was then centrifuged (275g) for 5 minutes at room temperature. The top layer of the sample wassiphoned off. Two milliliters of 0.1 N hydrochloric acid in 50%methanol/water was added, the mixture was centrifuged (275 g) and thetop layer was carefully siphoned off. The resulting liquid (thechloroform phase) was transferred to a liquid scintillation vial and airdried. Ten milliliters of a 2:1 (v/v) mixture of toluene and TritonX-100 containing 0.27% New England Nuclear Omnifluor was added, and theradioactivity was determined in a liquid scintillation counter.

Three replicates (samples of enzyme) were conducted per experiment (peranimal). The results were reported as the average of the values obtainedfrom the adipose tissue from two experiments (i.e., two differentanimals).

The percent inhibition by the test compound compared to the controls (notest compound) was determined.

The following individual species of the compounds of formula I weretested:

    ______________________________________                                        Compound                                                                      No.     Name                                                                  ______________________________________                                        1       N-(1-methyl-2-(4-methylphenyl)ethyl)-delta-phenyl-                            2-pyridinebutanamine, maleic acid salt                                2       N-(2-(4-chlorophenyl)-1,1-dimethylethyl)-delta-                               phenyl-2-pyridinebutanamine, maleic acid salt                         ______________________________________                                        The results were as follows:                                                                      Percent                                                   Compound No.        Inhibition                                                ______________________________________                                        1                   97                                                        2                   93                                                        ______________________________________                                    

The compounds of Formula I can be used to control triglyceridebiosynthesis in swine by administering an effective amount of one or amixture of two or more of the compounds orally or parenterally to theanimal. They may be administered as such, or as an active ingredient ofa conventional pharmaceutical formulation. They may be administeredorally by any convenient means. Thus, they may be orally administered asa drench, by intubation, in the animal's food and water, in a foodsupplement or in a formulation expressly designed for administration ofthe drug. Suitable formulations include solutions, suspensions,dispersions, emulsions, tablets, boluses, powders, granules, capsules,syrups and elixirs. For parenteral administration, they may be in theform of a solution, suspension, dispersion or emulsion. They can beadministered in the form of an implant or other controlled sustainedrelease formulation. Inert carriers, such as one or more of water,edible oil, gelatin, lactose, starch, magnesium stearate, talc orvegetable gum can be used. The dosage of the compound needed to inhibitsynthesis of the triglycerides will depend upon the particular compoundused, and the particular animal being treated. However, in general,satisfactory results are obtained when the compounds are administered ina dosage of from about 1 to about 500 milligrams per kilogram of theanimal's body weight. The compound can be administered in a single doseor in a series of doses in the same day, or over a period of days. Forany particular animal, a specific dosage regimen should be adjustedaccording to the individual need, the particular compound(s) used as theinhibitor, and the professional judgment of the person administering orsupervising the administration of the inhibitor. It is to be understoodthat the dosages set forth herein are exemplary only, and that they donot, to any extent, limit the scope or practice of the invention.

I claim:
 1. A method for inhibiting biosynthesis of triglycerides inswine which comprises administering, to a pig in need of such treatment,orally or parenterally, an effective amount of a compound of theformula: ##STR4## wherein (a) the bridging fragment >A--B-- is one of##STR5## and >C═CH--; (b) R is 2-pyridyl or ##STR6## (c) each of R¹ ishydrogen or methyl; (d) R² is hydrogen or hydroxyl;(e) n is zero or one;(f) X is halogen or alkyl of one to three carbon atoms; (g) m is zero orone; (h) Y is alkyl of one to three carbon atoms, and theirphysiologically acceptable acid addition salts.